Forensic sciences: Effects of stress and perturbations on soil communities/Introduction

Testate amoebae
Testate amoebae, thecamoebians, or testaceans are a polyphyletic group of unicellular ameboid protists. Under the current taxonomy, testate amoebas are classified in the group of amoeboid protozoans in the classes Lobosea and Filosea in the Superclass Rhizopoda.

Biology and ecology
They are found in many habitats such as mosses, soils, peatlands, lakes, rivers and estuarine environments around the world. In soils, the main factors explaining their abundance and community structure are the moisture content and water chemistry. Amoebae require the presence of humidity because, as aquatic organisms, a they have to live constantly in water. If conditions become less favorable, especially if the soil dries out, they have the ability to form cysts to avoid desiccation. Encystment in these species allows also dissemination. These protozoa can tolerate a wide temperature range depending on the species, thus they can be found in the tropics and in the polar regions. It has been also shown that the distribution of testate amoebae on earth is also limited by pH. Some species can live in acid moors and other in alkaline soils, while a smaller number of species can even tolerate both kinds of habitats.

As their name suggests, the feature of this group of amoebae is the presence of a test that is lacking in naked amoebae. This test is constituted by various elements.

Each species of testate amoeba has a specific test morphology and composition that allows to recognize and classify them. Even after the death of the amoebae, the tests remain in soils, sometimes for hundreds of years. As they are very sensitive to disturbances of environments they can be a good bioindicator in various research fields (ecology, paleoecology, paleolimnology, paleoclimatology, peatland regeneration, soil and air pollution monitoring and ecotoxicology). Their size varies between 10 and 500 micrometers. Most have a size between 20 and 200 micrometers but larger individuals have been observed with a size of up to 2000 micrometers.

Testate amoebae move and feed through its pseudopods. Smaller species are essentially bacterivorous. However larger species can also use other sources of food such as protists, including other testate amoebae or naked amoebae, fungi, small metazoans, algae and detrital organic particles. The reproduction mode, still little known, is by replication of the parent by asexual binary fission. The doubling time of the population, under natural conditions, is estimated between six and eleven days.

Classification
The two main criteria for classifying testate amoebas are the test structure and the pseudopod morphology. The type of test can be divided into three main groups according to its composition:
 * A protein test that is organic and made of composite structural proteins either blocks or homogenous layers.
 * An agglutinated test which comes from exogenous collected materials such as mineral particles of Diatoms or silica platelets of Euglyphida.
 * A siliceous or calcareous tests made of rods, nails or plates that are produced by the organisms themselves.

Testate amoebae can be separated into two groups according to the shape of their pseudopodia. Lobose testate amoebae are grouped within the phylum Amoebozoa and are characterized by lobed or finger-shape pseudopodia. Filose testate amoebae are grouped within the phylum Cercozoa and are characterized by thin pseudopodia. The development of new molecular methods molecular classifications now allows to reassessing the classification based on pseudopodia morphology.

Diversity
The two main testate amoebae orders are Arcellinida and Euglyphida. The order of Arcellinida belongs to the lobed testate amoebae and are the most numerous. They represent approximately three quarters of all known species of testate amoebae. The order of Euglyphida belongs to the filose testate amoebae.

Forensic sciences
Forensic sciences is the use of scientific knowledge and methodology in order to resolve criminal investigations. The estimation of the post-mortem interval (PMI) is one of the major purpose in forensic sciences because it is fundamental in solving criminal cases. Indeed, it allows, among other things, to restore order in which events occurred. Nowadays, there are two main possibilities to determine it: For a longer PMI, these techniques are no longer precise enough. Therefore, in the last decades, seeing that a cadaver alter visibly the surrounding environment, scientists have been interested in the influence of a cadaver on the soil components in order to discover new techniques allowing the PMI determination. Studies have been made on the effect of a cadaver on soil nutrients, pH and fungi and microbial communities. It has been shown that phosphorus, carbon and nitrogen concentrations (amoung others) as well as the pH level increases strongly in a soil below a cadaver. These enhancements persist two years after a corpse has been laid on the ground. Populations of bacteria and fungi also increase strongly in the presence of a cadaver.
 * 1) Medical techniques: These techniques allow the post mortem dating. It uses, for example, the temperature of the body or the rigor mortis. It allows to estimate the PMI from a couple of hours to approximately three days. After that amount of time, medical methods are no longer possible.
 * 2) Forensic entomology: By the observation of larval stages of flies and beetles, forensic entomologists are able to estimate the PMI after several weeks.

Therefore scientists have assumed that these major environmental changes beneath a cadaver could strongly influence the composition of testate amoebae communities, which are very sensitive, among others, to moisture rate, pH, nutrients and prey community changes.

Testate amoebae in forensic sciences
Testate amoebae is a group of protist which is frequently used as bioindicators. A bioindicator is an organism that can be used for the detection and quantitative characterization of a certain environmental factor or a complex of environmental factors. For example, testate amoebae seems to be the best agricultural bioindicator, as they are highly sensitive to pesticide treatments. Testate amoebae can be used as bioindicators, because they own characteristics that make them ideally suited to this type of study: A study interested in the influence of a cadaver on testate amoebae communities has shown that a cadaver strongly decrease the diversity and the abundance of testate amoebae beneath the body. Observations showed that after 22 to 33 days no living amoebae was still present under the corpse. A recovery of the amoebae community occured after the end of the active decay phase, which is from one month up to two months, but still after 10 months, recovery was not entirely completed.
 * 1) Their test is conserved even after the death of the organism and can even fossilize in peatlands (in other environments, the test is not conserved for a long time after death).
 * 2) They are present in a wide range of habitats and are very abundant.
 * 3) They are easy to find and identify (even if it is time-consuming and if the taxonomy isn't complete yet).
 * 4) They are strongly sensitive to abiotic and biotic environmental changes and their response can be observed very quickly, discernible by morphological adaptations and community structure changes.
 * 5) They can encyst and excyst under stressful circumstances.

The decrease may be caused by several motifs. An additional study showed that it was correlated with an important nitrogen and organic carbon input in the soil beneath the cadaver. The decrease might occured because of the anoxic conditions or because of the direct high nitrogen concentration. Furthermore, the large nutrients input and pH variations (caused by the intake of ammonium ions ) produces big changes in the bacterial abundance and community structure. Bacteria are the main diet of testate amoebae and it is likely that some species are very specialized in a type of prey. It is therefore possible that any change in the prey community results in a change in the amoebae community. Indeed, it has been proven in several studies that testate amoebae are strongly linked to moisture rate, pH, temperature, prey availability, and nutrients changes, by direct or indirect effects. The changes that occur in community structure, following a chronological pattern, could be used as a tool to determine the PMI.

In conclusion, using testate amoebae for forensic purposes, with a focus on testate amoebae community structure, recovery time and succession pattern, is totally appopriate.

Even if the taxonomy is still incomplete and the species identification time-consuming, improvements in molecular approaches are likely to make the use of testate amoebaes in forensic sciences more accessible.

A cadaver in the ecosystem
The decomposition process is a central point in the energy and matter cycles. Indeed,  Philip S. Barton wrote:

''« Up to 90 % of organic matter generated by plants is not consumed when living and enters the detritus pool. Animals consume the remaining 10 %, incorporate this into new tissue for growth and development, and eventually return these nutrients to the detritus pool as excreta and carrion”''.

The arrival of a cadaver in an ecosystem leads to important changes. Indeed, its fast decomposition process, its nutrient richness and its patchy influence make him a fundamental hotspot of biological and chemical activity (compared to plants).

Furthermore, it brings a large increase in the biodiversity, caused by the attraction of insects, microorganisms, scavengers and predators for this unusual source of food. A carrion decomposition, controlled by temperature and moisture rate, affects and accelerates the temporal and the spatial dynamic of the ecosystem and its communities. The spatial dynamic is influenced by the energy and nutrients flows and the temporal dynamic is influenced by the species succession.

The microbes are fundamental in the recycling process because they mineralize the fundamental nutrients and make them available for plants, which are the initial link of the food chain. Arthropods and scavengers disperse the nutrients in an horizontal direction. At the end, all animal, predator or prey, will re-enter the decomposition cycle upon its death.

Ecological succession theory
Ecological succession is the observed process of change in the species structure of an ecological community over time. The time scale can be decades (for example, after a wildfire), or even millions of years after a mass extinction. For a cadaver, it can be counted in days.

The community begins with relatively few pioneering plants and animals and develops through increasing complexity until it becomes stable or self-perpetuating as a climax community. The ʺengineʺ of succession, the cause of ecosystem change, is the impact of established species upon their own environments. A consequence of living is the sometimes subtle and sometimes overt alteration of one's own environment.

The difference between a succession in plant ecology and in carrion ecology is that once the resource is exhausted, the carrion ecosystem does not reach a climax. Nevertheless, lots of studies have demonstrated that different communities of organisms (especially arthropods) follow each other through time, in parallel with the progress of the various decomposition stages (The composition of the cadaver changes quickly during the decomposition process, providing various sort of food.). It allows, for example, the use of insects to evaluate the PMI. It is possible that this theory could entirely be applicated to testate amoebae.

Cadaver impact on soil nutrients, moisture, pH and bacteria and fungal communities
A cadaver has a fast, huge and localised impact on soil also called « Cadaver Decomposition Island (CDI) ». It releases a large amount of nutrients like nitrogen, carbon or phosphorous. The release of ammonium ions leads to an increasement of the pH. This changes follow a predictable temporal pattern correlated with the mass loss of the cadaver. The release of body fluids and its nutrients in the soil strongly increases the microbial biomass and activity. Indeed, fungal and bacterial abundance and diversity are directly linked with the nutrient content of the soil. Furthermore, in the edges of the CDI,  the soil is more fertile and has a broader biodiversity and biomass production.

Decomposition stages
Five general stages are used to describe the process of decomposition in vertebrate animals: fresh, bloat, active and advanced decay, and dry/remains. The general stages of decomposition are coupled with two stages of chemical decomposition: autolysis and putrefaction. These two stages contribute to the chemical process of decomposition, which breaks down the main components of the body.
 * 1) The “fresh” stage starts at the cessation of the heart and depletion of internal oxygen. Without oxygen, the aerobic metabolism stops and the enzymatic digestion of cells (autolysis) starts. The flies lay eggs and the maggot starts to eat the tissues. Bacterial populations grow up within 24h.
 * 2) The “bloated” stage is caused, after 48 hours, by the lack of oxygen which promotes the anaerobic bacteria, which transform sugar and lipids into organic acids like propionic or lactic acid. This is the putrefaction stage. The gas pressure push the fluids out of the body by the orifices (mouth, nose, anus).
 * 3) The “active decay” stage starts when seepage and maggots cause rips in the body skin, which make oxygen come back and maggot and aerobic activity increases again. At this stage, the mass loss is huge.
 * 4) The “advanced decay” stage occurs with the transformation of maggots into pupae. At this stage, the increase of carbon, nutrient and pH of the soil is not linked with a positive effect on soil biology. Indeed, the vegetation and most organisms like collembolan and acari die for many reasons like nitrogen toxicity and suffocation.
 * 5) The “dry” and “remains” stages are the last stages of the decomposition process. The “dry” stage is characterized by an increase of vegetation around the CDI. The “remains” stage is characterized by an increase of vegetation inside the CDI.  The nutrients and moisture are depleted quickly, because easily accessible, but the concentrations of nutrients in soil doesn’t decrease. Collembolan and acari populations decrease and fungi form fruiting structures due to the high nitrogen level.

Purposes
Further to ancient studies on the impact of a cadaver on testate amoebae soil communities, our study has to verify if the observed variations are really due to the cadaver in its entirety or to its body fluids only. Thus the study will compare the effect of pig cadaver, pig blood and cow manure. Blood and manure can be important in the determination of the PMI because if the body is missing, they are the only elements that remain at the crime scene. Furthermore, their impact on testate amoebae communities is still unknown. The final aims of this study are to confirm that using testate amoebae in forensic sciences is founded and to broaden the scarce knowledge in this new scientific field.

Realization of the experiment
To achieve our purposes, three treatments will be set up and applicated to soil and litter: pigs heads, pig blood, and cow manure. A control composed only of soil and litter will be set up too. The follow-up of treatments lasts four weeks, and sampling days take place once per week (so in total four times).

The pigs heads treatment has been imagined in order to imitate the cadaver impacts son soil, although the blood and manure allow to test body fluids impacts.

To analyze the treatments impacts on soil communities, three parameters will be measured: respirometry, pH and the species diversity and relative abundance of living and dead testate amoebae (living/dead ratio).
 * The species diversity and the relative abundance of living and dead testate amoebae are naturally analyzed because these organisms respond very well to disturbances, implying that a change in the community can be used as a tool for the PMI determination.
 * The respirometry allows to measure the metabolic activity of the soil organisms. An increase in the CO2 flow measured by the respirometer means an increase in the soil metabolic activity, but not necessary an increase in testate amoebae community. These data could help to determine a potential correlation between an increase of the soil metabolic activity (bacteria, fungi...) and an increase in amoebae abundance. Indeed, testate amoebae eat bacteria and fungi and an increase in the prey abundance should usually increases the predator abundance, but testate amoebae being very sensitive to a change of prey or abiotic environment, this will not necessarily be observed.
 * The pH is measured because testate amoebae are very sensitive to this so a change in the pH should be correlated to a change in the amoebae community. This would confirm previous studies.

Hypothesis
Testate amoebae being very versatils creatures, their short reproductive cycle should allow to observe evolutions of populations through the four treatments and time. They are sensitive to dryness and pH. Thus, different panels of amoebae species and different living/dead ratio are expected to be found in each treatment pot through time.

The three treatments should influence differently moisture and pH, which are both determining factors of testate amoebae communities structure. Indeed, an increase in the moisture rate should increase the amoebae species diversity, because they live in the soil water and depend on it. Contrariwise, a variation of the pH should decrease the amoebae species diversity because they are strongly "pH-dependant". The pH and the moisture rate should have direct and an indirect influences. The direct influences may be caused by a change in the abiotic conditions and the indirect influences may be caused by changes in species diversity of prey community. The presence of the pig head and the cow manure on the litter should cause a higher soil humidity rate than in control and blood treatments, which should lead to an increase in living testate amoebae species diversity. The presence of the pig head and blood should increase the pH to more basic conditions, which should lead to a decrease in living testate amoebae species diversity. The manure should decrease the pH to more acidic conditions, which should also lead to a decrease in living testate amoebae species diversity.

The nutrient content of the soil also has a huge impact on soil communities. An increase in nutrients such as carbon, nitrogen, or phosphorous could drastically change the soil communities composition, by, for example totally modify the bacterial flora composition. Indeed, the nutrients can have an impact because of their role of nutritive element but also because they can change the pH (ammonium ions for example) or be toxic (too much nitrate for example).

According to these predictions, the control treatment samples should be the driest and have the smallest pH variations. Thus the testate ameobae community shouldn't change through time, except because of dry or wet weather conditions.

The cow manure treatment samples should be a little more wet because the dung forms a waterproof layer and keeps the humidity in the litter. Through this we expect to see an acidification of the soil and the acidophilic amoebae species, such as Arcella sp., Assulina muscorum, Corythion dubium, Difflugia lucida, Euglypha sp., Nebela collaris and Nebela tincta, should become more abundant. The manure decomposition process is quiet long so its influence on the nutrient content of the soil should not be seen the first two weeks. Then the nutrient introduction in the soil by the decomposers should change the prey communities and decrease the species diversity of living testate amoebae.

The blood treatment samples should be as dry as the control ones but the blood should affect the pH and the nutrient content of the soil, so some communities evolution should be visible. The changes in prey communities should negatively affect the species diversity of the living testate amoebae community, which are quite specific predators.

The pig head treatment samples should be affected by pH variations. The first two weeks, the pH should increase and reach a slightly basic level. Then it should go back to a neutral level. This should affect the amoebae communities and allow basidophilic species to spread, such as Centropyxis aerophila, Nebela bohemica and Nebela lageniformis. The moisture level should be higher than in the other samples because of the influence of cadaveric fluids and the head shadow. It should be positive for the reproduction rate. The nutrient content of the soil should increase and have a negativ impact on the living testate amoebae community, like for the blood treatment.